Methods of treatment using a JAK inhibitor compound

ABSTRACT

The invention relates to methods of treating ocular diseases and certain respiratory diseases using the compound 5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol 
                         
or a pharmaceutically-acceptable salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.16/525,859, filed on Jul. 30, 2019, now allowed, which is a divisionalapplication of U.S. Ser. No. 15/966,438, filed on Apr. 30, 2018, nowU.S. Pat. No. 10,406,148, which application claims the benefit of U.S.Provisional Application No. 62/492,568, filed on May 1, 2017, thedisclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to methods for treating ocular andcertain respiratory diseases using a particular JAK inhibitor compoundor a pharmaceutically-acceptable salt thereof.

State of the Art

Cytokines are intercellular signaling molecules which includechemokines, interferons, interleukins, lymphokines, and tumour necrosisfactor. Cytokines are critical for normal cell growth andimmunoregulation but also drive immune-mediated diseases and contributeto the growth of malignant cells. Elevated levels of many cytokines havebeen implicated in the pathology of a large number of diseases orconditions, particularly those diseases characterized by inflammation.Many of the cytokines implicated in disease act through signalingpathways dependent upon the Janus family of tyrosine kinases (JAKs),which signal through the Signal Transducer and Activator ofTranscription (STAT) family of transcription factors.

The JAK family comprises four members, JAK1, JAK2, JAK3, and tyrosinekinase 2 (TYK2). Binding of cytokine to a JAK-dependent cytokinereceptor induces receptor dimerization which results in phosphorylationof tyrosine residues on the JAK kinase, effecting JAK activation.Phosphorylated JAKs, in turn, bind and phosphorylate various STATproteins which dimerize, internalize in the cell nucleus and directlymodulate gene transcription, leading, among other effects, to thedownstream effects associated with inflammatory disease. The JAKsusually associate with cytokine receptors in pairs as homodimers orheterodimers. Specific cytokines are associated with specific JAKpairings. Each of the four members of the JAK family is implicated inthe signaling of at least one of the cytokines associated withinflammation.

Inflammation plays a prominent role in many ocular diseases, includinguveitis, diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, and atopic keratoconjunctivitis.Uveitis encompasses multiple intraocular inflammatory conditions and isoften autoimmune, arising without a known infectious trigger. Thecondition is estimated to affect about 2 million patients in the US. Insome patients, the chronic inflammation associated with uveitis leads totissue destruction, and it is the fifth leading cause of blindness inthe US. Cytokines elevated in uveitis patients' eyes that signal throughthe JAK-STAT pathway include IL-2, IL-4, IL-5, IL-6, IL-10, IL-23, andIFN-γ. (Horai and Caspi, J Interferon Cytokine Res, 2011, 31, 733-744;Ooi et al, Clinical Medicine and Research, 2006, 4, 294-309). Existingtherapies for uveitis are often suboptimal, and many patients are poorlycontrolled. Steroids, while often effective, are associated withcataracts and increased intraocular pressure/glaucoma.

Diabetic retinopathy (DR) is caused by damage to the blood vessels inthe retina. It is the most common cause of vision loss among people withdiabetes. Angiogenic as well as inflammatory pathways play an importantrole in the disease. Often, DR will progress to diabetic macular edema(DME), the most frequent cause of visual loss in patients with diabetes.The condition is estimated to affect about 1.5 million patients in theUS alone, of whom about 20% have disease affecting both eyes. Cytokineswhich signal through the JAK-STAT pathway, such as IL-6, as well asother cytokines, such as IP-10 and MCP-1 (alternatively termed CCL2),whose production is driven in part by JAK-STAT pathway signaling, arebelieved to play a role in the inflammation associated with DR/DME(Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12; Sohn et al.,American Journal of Opthalmology, 2011, 152, 686-694; Owen and Hartnett,Curr Diab Rep, 2013, 13, 476-480; Cheung et al, Molecular Vision, 2012,18, 830-837; Dong et al, Molecular Vision, 2013, 19, 1734-1746; Funatsuet al, Ophthalmology, 2009, 116, 73-79). The existing therapies for DMFare suboptimal: intravitreal anti-VEGF treatments are only effective ina fraction of patients and steroids are associated with cataracts andincreased intraocular pressure.

Dry eye disease (DED) is a multifactorial disorder that affectsapproximately 5 million patients in the US. Ocular surface inflammationis believed to play an important role in the development and propagationof this disease. Elevated levels of cytokines such as IL-1, IL-2, IL-4,IL-5, IL-6, and IFN-γ have been noted in the ocular fluids of patientswith DED, (Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and thelevels often correlated with disease severity. Age-related maculardegeneration and atopic keratoconjunctivitis are also thought to beassociated with JAK-dependent cytokines.

Given the number of cytokines elevated in inflammatory diseases and thateach cytokine is associated with a particular JAK pairing, it would bedesirable to provide a chemical inhibitor with pan-activity against allmembers of the JAK family for the treatment of ocular disease. However,the broad anti-inflammatory effect of such inhibitors could suppressnormal immune cell function, potentially leading to increased risk ofinfection. It would be desirable, therefore, to provide an inhibitorthat can be locally delivered to the site of action in the eye, therebylimiting the potential for adverse systemic immunosuppression.

Commonly assigned U.S. application Ser. No. 15/341,226, filed Nov. 2,2016 discloses diamino compounds useful as JAK inhibitors. Inparticular, the compound5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(compound 1)

is specifically disclosed in the application as a potent pan-JAKinhibitor. This application discloses various uses of compound 1, inparticular treatment of respiratory diseases including asthma, chronicobstructive pulmonary disease, cystic fibrosis, pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema and bronchiolitis obliterans. However, this application doesnot disclose the use of compound 1 for the treatment of ocular disease.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating ocular diseases orsymptoms thereof using the JAK inhibitor5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolor a pharmaceutically-acceptable salt thereof.

In one aspect, the invention provides a method of treating an oculardisease in a human patient, the method comprising administering to theeye of the patient, the compound5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolof formula

hereinafter compound 1, or a pharmaceutically-acceptable salt thereof.

In one aspect the ocular disease is uveitis, diabetic retinopathy,diabetic macular edema, dry eye disease, age-related maculardegeneration, or atopic keratoconjunctivitis. In particular, the oculardisease is uveitis or diabetic macular edema.

In another aspect, the invention provides a pharmaceutical compositionof5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(compound 1) or a pharmaceutically-acceptable salt thereof wherein thepharmaceutical composition is suitable for administration directly tothe eye of a patient.

The present invention further relates to methods of using compound 1 totreat certain specific respiratory diseases.

In one aspect, the invention provides a method of treating a respiratorydisease in a mammal, the method comprising administering to the mammal apharmaceutical composition comprising compound 1 or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically-acceptable carrier,wherein the respiratory disease is a lung infection, a helminthicinfection, pulmonary arterial hypertension, sarcoidosis,lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonarydisease.

In yet another aspect, the invention provides a method of treating arespiratory disease in a mammal, the method comprising administering tothe mammal a pharmaceutical composition comprising compound 1 or apharmaceutically acceptable salt thereof, and apharmaceutically-acceptable carrier, wherein the respiratory disease isdrug-induced pneumonitis, fungal induced pneumonitis, allergicbronchopulmonary aspergillosis, hypersensitivity pneumonitis,eosinophilic granulomatosis with polyangiitis, idiopathic acuteeosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia,hypereosinophilic syndrome, Löffler syndrome, bronchiolitis obliteransorganizing pneumonia, or immune-checkpoint-inhibitor inducedpneumonitis.

DETAILED DESCRIPTION OF THE INVENTION

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).

Furthermore, the imidazo portion of the tetrahydroimidazopyridine moietyin the structure of the present compound exists in tautomeric forms. Thecompound could equivalently be represented as

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the imidazopyridine portion.Accordingly this structure is designated5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.It will be understood that although structures are shown, or named, in aparticular form, the invention also includes the tautomer thereof.

Definitions

When describing the present invention, the following terms have thefollowing meanings unless otherwise indicated.

The singular terms “a,” “an” and “the” include the corresponding pluralterms unless the context of use clearly dictates otherwise.

The term “about” means±5 percent of the specified value.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment,e.g., the amount needed to obtain the desired therapeutic effect.

The term “treating” or “treatment” means preventing, ameliorating orsuppressing the medical condition, disease or disorder being treated(e.g., a respiratory disease) in a patient (particularly a human); oralleviating the symptoms of the medical condition, disease or disorder.

The term “unit dosage form” or “unit doses” means a physically discreteunit suitable for dosing a patient, i.e., each unit containing apredetermined quantity of a therapeutic agent calculated to produce atherapeutic effect either alone or in combination with one or moreadditional units. Examples include capsules, tablets and the like.

All other terms used herein are intended to have their ordinary meaningas understood by persons having ordinary skill in the art to which theypertain.

The term “pharmaceutically-acceptable” means acceptable foradministration to a patient (e.g., having acceptable safety for thespecified usage).

The term “pharmaceutically-acceptable salt” means a salt prepared froman acid and a base (including zwitterions) that is acceptable foradministration to a patient (e.g., a salt having acceptable safety for agiven dosage regime).

Representative pharmaceutically acceptable salts include salts ofacetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, oxalic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like.

Compound 1

The present method invention employs5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(compound 1)

or a pharmaceutically-acceptable salt thereof.

Compound 1 may be prepared as described in U.S. application Ser. No.15/341,226 or in the appended examples.

In one aspect of the invention, compound 1 is employed in the form of acrystalline freebase hydrate characterized by a powder X-ray diffraction(PXRD) pattern having significant diffraction peaks, among other peaks,at 20 values of 6.20±0.20, 9.58±0.20, 17.53±0.20, 19.28±0.20, and21.51±0.20. The preparation of the crystalline hydrate is also describedin U.S. Ser. No. 15/341,226 and in the examples below.

Pharmaceutical Compositions

The present compound,5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(1) and pharmaceutically-acceptable salts thereof is typically used inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may advantageously be administered to apatient by any acceptable route of administration including, but notlimited to, oral, inhalation, optical injection, topical (includingtransdermal), rectal, nasal, and parenteral modes of administration.

The pharmaceutical compositions utilized in the invention typicallycontain a therapeutically effective amount of compound 1. Those skilledin the art will recognize, however, that a pharmaceutical compositionmay contain more than a therapeutically effective amount, i.e., bulkcompositions, or less than a therapeutically effective amount, i.e.,individual unit doses designed for multiple administration to achieve atherapeutically effective amount. When discussing compositions and uses,compound 1 may also be referred to herein as ‘active agent’.

Typically, pharmaceutical compositions will contain from about 0.01 toabout 95% by weight of the active agent; including, for example, fromabout 0.05 to about 30% by weight; and from about 0.1% to about 10% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions utilized in the invention. The choice of a particularcarrier or excipient, or combinations of carriers or excipients, willdepend on the mode of administration being used to treat a particularpatient or type of medical condition or disease state. In this regard,the preparation of a suitable pharmaceutical composition for aparticular mode of administration is well within the scope of thoseskilled in the pharmaceutical arts. Additionally, the carriers orexcipients used in the pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

In one aspect, the pharmaceutical composition is suitable for ocularinjection. In this aspect, the compound may be formulated as a sterileaqueous suspension or solution. Useful excipients that may be includedin such an aqueous formulation include polysorbate 80,carboxymethylcellulose, potassium chloride, calcium chloride, magnesiumchloride, sodium acetate, sodium citrate, histidine, α-α-trehalosedihydrate, sucrose, polysorbate 20, hydroxypropyl-β-cyclodextrin, andsodium phosphate. Benzyl alcohol may serve as a preservative and sodiumchloride may be included to adjust tonicity. In addition, hydrochloricacid and/or sodium hydroxide may be added to the solution for pHadjustment. Aqueous formulations for ocular injection may be prepared aspreservative-free.

In another aspect, the pharmaceutical composition is suitable forinhaled administration. Pharmaceutical compositions for inhaledadministration are typically in the form of an aerosol or a powder. Suchcompositions are generally administered using inhaler delivery devices,such as a dry powder inhaler (DPI), a metered-dose inhaler (MDI), anebulizer inhaler, or a similar delivery device.

In a particular embodiment, the pharmaceutical composition isadministered by inhalation using a dry powder inhaler. Such dry powderinhalers typically administer the pharmaceutical composition as afree-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free-flowing powder composition, thetherapeutic agent is typically formulated with a suitable excipient suchas lactose, starch, mannitol, dextrose, polylactic acid (PLA),polylactide-co-glycolide (PLGA) or combinations thereof. Typically, thetherapeutic agent is micronized and combined with a suitable carrier toform a composition suitable for inhalation.

A representative pharmaceutical composition for use in a dry powderinhaler comprises lactose and the compound of the invention inmicronized form. Such a dry powder composition can be made, for example,by combining dry milled lactose with the therapeutic agent and then dryblending the components. The composition is then typically loaded into adry powder dispenser, or into inhalation cartridges or capsules for usewith a dry powder delivery device.

Dry powder inhaler delivery devices suitable for administeringtherapeutic agents by inhalation are described in the art and examplesof such devices are commercially available. For example, representativedry powder inhaler delivery devices or products include Aeolizer(Novartis); Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler(GlaxoSmithKiine); Diskus/Accuhaler (GlaxoSmithKline); Ellipta(GlaxoSmithKline); Easyhaler (Orion Pharma); Eclipse (Aventis); FlowCaps(Hovione); Handihaler (Boehringer Ingelheim); Pulvinal (Chiesi);Rotahaler (GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma);Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler(Aventis); and the like.

In another particular embodiment, the pharmaceutical composition isadministered by inhalation using a metered-dose inhaler. Suchmetered-dose inhalers typically discharge a measured amount of atherapeutic agent using a compressed propellant gas. Accordingly,pharmaceutical compositions administered using a metered-dose inhalertypically comprise a solution or suspension of the therapeutic agent ina liquefied propellant. Any suitable liquefied propellant may beemployed including hydrofluoroalkanes (HFAs), such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227); and chlorofluorocarbons,such as CCl₃F. In a particular embodiment, the propellant ishydrofluoroalkanes. In some embodiments, the hydrofluoroalkaneformulation contains a co-solvent, such as ethanol or pentane, and/or asurfactant, such as sorbitan trioleate, oleic acid, lecithin, andglycerin.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of the compoundof the invention; from about 0% to about 20% by weight ethanol; and fromabout 0% to about 5% by weight surfactant; with the remainder being anHFA propellant. Such compositions are typically prepared by addingchilled or pressurized hydrofluoroalkane to a suitable containercontaining the therapeutic agent, ethanol (if present) and thesurfactant (if present). To prepare a suspension, the therapeutic agentis micronized and then combined with the propellant. The composition isthen loaded into an aerosol canister, which typically forms a portion ofa metered-dose inhaler device.

Metered-dose inhaler devices suitable for administering therapeuticagents by inhalation are described in the art and examples of suchdevices are commercially available. For example, representativemetered-dose inhaler devices or products include AeroBid Inhaler System(Forest Pharmaceuticals); Atrovent Inhalation Aerosol (BoehringerIngelheim); Flovent (GlaxoSmithKline); Maxair Inhaler (3M); ProventilInhaler (Schering); Serevent Inhalation Aerosol (GiaxoSmithKline); andthe like.

In another particular aspect, the pharmaceutical composition isadministered by inhalation using a nebulizer inhaler. Such nebulizerdevices typically produce a stream of high velocity air that causes thepharmaceutical composition to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the therapeutic agent can be dissolved in a suitablecarrier to form a solution. Alternatively, the therapeutic agent can bemicronized or nanomilled and combined with a suitable carrier to form asuspension.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises a solution or suspension comprising from about 0.05μg/mL to about 20 mg/mL of the compound of the invention and excipientscompatible with nebulized formulations. In one embodiment, the solutionhas a pH of about 3 to about 8.

Nebulizer devices suitable for administering therapeutic agents byinhalation are described in the art and examples of such devices arecommercially available. For example, representative nebulizer devices orproducts include the Respimat Softmist Inhaler (Boehringer Ingelheim);the AERx Pulmonary Delivery System (Aradigm Corp.); the PARI LC PlusReusable Nebulizer (Pari GmbH); and the like.

In yet another aspect, the pharmaceutical compositions of the inventionmay alternatively be prepared in a dosage form intended for oraladministration. Suitable pharmaceutical compositions for oraladministration may be in the form of capsules, tablets, pills, lozenges,cachets, dragees, powders, granules; or as a solution or a suspension inan aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oilliquid emulsion; or as an elixir or syrup; and the like; each containinga predetermined amount of a compound of the present invention as anactive ingredient.

When intended for oral administration in a solid dosage form, thepharmaceutical compositions of the invention will typically comprise theactive agent and one or more pharmaceutically-acceptable carriers, suchas sodium citrate or dicalcium phosphate. Optionally or alternatively,such solid dosage forms may also comprise: fillers or extenders,binders, humectants, solution retarding agents, absorption accelerators,wetting agents, absorbents, lubricants, coloring agents, and bufferingagents. Release agents, wetting agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the pharmaceutical compositions of the invention.

Alternative formulations may also include controlled releaseformulations, liquid dosage forms for oral administration, transdermalpatches, and parenteral formulations. Conventional excipients andmethods of preparation of such alternative formulations are described,for example, in the reference by Remington, supra.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Aqueous Formulation for Ocular Injection

Each mL of a sterile aqueous suspension includes from 5 mg to 50 mg ofcompound 1, sodium chloride for tonicity, 0.99% (w/v) benzyl alcohol asa preservative, 0.75% carboxymethylcellulose sodium, and 0.04%polysorbate. Sodium hydroxide or hydrochloric acid may be included toadjust pH to 5 to 7.5.

Aqueous Formulation for Ocular Injection

A sterile preservative-free aqueous suspension includes from 5 mg/mL to50 mg/mL of compound 1 in 10 mM sodium phosphate, 40 mM sodium chloride,0.03% polysorbate 20, and 5% sucrose.

Dry Powder Composition

Micronized compound 1 (1 g) is blended with milled lactose (2.5 g). Thisblended mixture is then loaded into individual blisters of a peelableblister pack in an amount sufficient to provide between about 0.1 mg toabout 4 mg of the compound of formula I per dose. The contents of theblisters are administered using a dry powder inhaler.

Dry Powder Composition

Micronized compound 1 (1 g) is blended with milled lactose (20 g) toform a bulk composition having a weight ratio of compound to milledlactose of 1:20. The blended composition is packed into a dry powderinhalation device capable of delivering between about 0.1 mg to about 4mg of the compound of formula I per dose.

Metered-Dose Inhaler Composition

Micronized compound 1 (10 g) is dispersed in a solution prepared bydissolving lecithin (0.2 g) in demineralized water (200 mL). Theresulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded intometered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 0.1mg to about 4 mg of the compound of formula I per dose when administeredby the metered dose inhaler.

Nebulizer Composition

Compound 1 (25 mg) is dissolved in a solution containing 1.5-2.5equivalents of hydrochloric acid, followed by addition of sodiumhydroxide to adjust the pH to 3.5 to 5.5 and 3% by weight of glycerol.The solution is stirred well until all the components are dissolved. Thesolution is administered using a nebulizer device that provides about0.1 mg to about 4 mg of the compound of formula I per dose.

Utility

The present compound,5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,(compound 1), has been shown to be a potent inhibitor of the JAK familyof enzymes: JAK1, JAK2, JAK3, and TYK2.

Ocular Diseases

Many ocular diseases have been shown to be associated with elevations ofproinflammatory cytokines that rely on the JAK-STAT pathway. Since thecompound of the invention exhibits potent inhibition at all four JAKenzymes, it is expected to potently inhibit the signaling and pathogeniceffects of numerous cytokines (such as IL-6, IL-2 and IFN-γ), thatsignal through JAK, as well as to prevent the increase in othercytokines (such as MCP-1 and IP-10), whose production is driven byJAK-STAT pathway signaling.

In particular, the present compound exhibited pIC₅₀ values of 6.7 orgreater (IC₅₀ values of 200 nM or less) for inhibition of IL-2, IL-4,IL-6, and IFNγ signaling in the cellular assays described in Assays 3 to7, including assays registering inhibition of the downstream effects ofcytokine elevation.

The pharmacokinetic study of Assay 8 demonstrated sustained exposure inrabbit eyes after a single intravitreal injection and a concentration inplasma at least three orders of magnitude lower than that observed invitreous tissue.

Furthermore, intravitreal dosing of the compound of the invention hasdemonstrated significant inhibition of IL-6 induced pSTAT3 in the ratretina/choroid tissue as well as significant inhibition of IFN-γ inducedIP-10 in the rabbit vitreous as well as retina/choroid tissues.

It is expected that sustained ocular JAK inhibition in the absence ofsignificant systemic levels will result in potent, localanti-inflammatory activity in the eye without systemically-drivenadverse effects. The compound of the invention is expected to bebeneficial in a number of ocular diseases that include, but are notlimited to, uveitis, diabetic retinopathy, diabetic macular edema, dryeye disease, age-related macular degeneration, and atopickeratoconjunctivitis.

In particular, uveitis (Horai and Caspi, J Interferon Cytokine Res,2011, 31, 733-744), diabetic retinopathy (Abcouwer, J Clin Cell Immunol,2013, Suppl 1, 1-12), diabetic macular edema (Sohn et al., AmericanJournal of Opthalmology, 201.1, 152, 686-694), dry eye disease(Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and age-relatedmacular degeneration (Knickelbein et al, Int Ophthalmol Clin, 2015,55(3), 63-78) are characterized by elevation of certain pro-inflammatorycytokines that signal via the JAK-STAT pathway. Accordingly, compoundsof the invention may be able to alleviate the associated ocularinflammation and reverse disease progression or provide symptom relief.

Retinal vein occlusion (RVO) is a highly prevalent visually disablingdisease. Obstruction of retinal blood flow can lead to damage of theretinal vasculature, hemorrhage, and tissue ischemia. Although thecauses for RVO are multifactorial, both vascular as well as inflammatorymediators have been shown to be important (Deobhakta et al,International Journal of Inflammation, 2013, article ID 438412).Cytokines which signal through the JAK-STAT pathway, such as IL-6 andIL-13, as well as other cytokines, such as MCP-1, whose production isdriven in part by JAK-STAT pathway signaling, have been detected atelevated levels in ocular tissues of patients with RVO (Shchuko et al,Indian Journal of Ophthalmology, 2015, 63(12), 905-911). Accordingly,compound 1 may be able to alleviate the associated ocular inflammationand reverse disease progression or provide symptom relief in thisdisease. While many patients with RVO are treated by photocoagulation,this is an inherently destructive therapy. Anti-VEGF agents are alsoused, but they are only effective in a fraction of patients. Steroidmedications that reduce the level of inflammation in the eye(Triamcinolone acetonide and dexamethasone implants) have also beenshown to provide beneficial results for patients with certain forms ofRVO, but they have also been shown to cause cataracts and increasedintraocular pressure/glaucoma.

In one aspect, therefore, the invention provides a method of treating anocular disease in a mammal, the method comprising administering5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolor a pharmaceutically-acceptable salt thereof to the eye of the mammal.In one aspect, the ocular disease is uveitis, diabetic retinopathy,diabetic macular edema, dry eye disease, age-related maculardegeneration, retinal vein occlusion, or atopic keratoconjunctivitis. Inone aspect, the method comprises administering the present compound byintravitreal injection.

Respiratory Diseases

The present compound,5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(1) has demonstrated inhibition of T cell activation, inhibition ofcytokines associated with inflammation, and activity in rodent lungeosinophilia and neutrophilia assays. Therefore, the compound isbelieved to be useful for the treatment of certain specific respiratorydiseases.

Eosinophilic airway inflammation is a characteristic feature of diseasescollectively termed eosinophilic lung diseases (Coffin et al., Clin.Chest. Med., 2016, 37(3), 535-56). Eosinophilic diseases have beenassociated with IL-4, IL-13 and IL-5 signaling. Eosinophilic lungdiseases include infections (especially helminthic infections),drug-induced pneumonitis (induced for example by therapeutic drugs suchas antibiotics, phenytoin, or 1-tryptophan), fungal-induced pneumonitis(e.g. allergic bronchopulmonary aspergillosis), hypersensitivitypneumonitis and eosinophilic granulomatosis with polyangiitis (formerlyknown as Churg-Strauss syndrome). Eosinophilic lung diseases of unknownetiology include idiopathic acute eosinophilic pneumonia, idiopathicchronic eosinophilic pneumonia, hypereosinophilic syndrome, and Löfflersyndrome. Compound 1 has been shown to significantly reduce lungeosinophilia in the rodent airway model of Assay 13 and to potentlyinhibit IL-13, IL-4, and IL-2 signaling in cellular assays.

A polymorphism in the IL-6 gene has been associated with elevated IL-6levels and an increased risk of developing pulmonary arterialhypertension (PAH) (Fang et al., J Am Soc Hypertens.,Interleukin-6-572C/G polymorphism is associated with serum interleukin-6levels and risk of idiopathic pulmonary arterial hypertension, 2017,ahead of print). Corroborating the role of IL-6 in PAH, inhibition ofthe IL-6 receptor chain gp130 ameliorated the disease in a rat model ofPAH (Huang et al., Can J Cardiol., 2016, 32(11), 1356.e1-1356.e10). Thecompound of the invention has been shown to inhibit IL-6 signaling.

Cytokines such as IFNγ, IL-12 and IL-6 have been implicated in a rangeof non-allergic lung diseases such as sarcoidosis, andlymphangioleiomyomatosis (El-Hashemite et al., Am. Respir. Cell Mol.Biol., 2005, 33, 227-230, and El-Hashemite et al., Cancer Res., 2004,64, 3436-3443). The compound of the invention has also been shown toinhibit IL-6 and IFNγ signaling.

Bronchiectasis and infiltrative pulmonary diseases are diseasesassociated with chronic neutrophilic inflammation. The compound of theinvention has been shown to inhibit airway neutrophilia in a rodentmodel.

Pathological T cell activation is critical in the etiology of multiplerespiratory diseases. Autoreactive T cells play a role in bronchiolitisobliterans organizing pneumonia (also termed COP [cryptogenic organizingpneumonia]). More recently, immune-checkpoint inhibitor inducedpneumonitis, another T cell mediated lung disease emerged with theincreased use of immune-checkpoint inhibitors. In cancer patientstreated with these T cell stimulating agents, fatal pneumonitis candevelop. The compound of the invention has been shown to inhibitactivation in T cells isolated from human peripheral blood mononuclearcells.

In one aspect, therefore, the invention provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolor a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier and thecompound of the invention, or a pharmaceutically acceptable saltthereof, wherein the respiratory disease is a lung infection, ahelminthic infection, pulmonary arterial hypertension, sarcoidosis,lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonarydisease.

In another aspect, the invention provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal the compound of the invention, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier and thecompound of the invention, or a pharmaceutically acceptable saltthereof, wherein the respiratory disease is drug-induced pneumonitis,fungal induced pneumonitis, allergic bronchopulmonary aspergillosis,hypersensitivity pneumonitis, eosinophilic granulomatosis withpolyangiitis, idiopathic acute eosinophilic pneumonia, idiopathicchronic eosinophilic pneumonia, hypereosinophilic syndrome, Löfflersyndrome, bronchiolitis obliterans organizing pneumonia, orimmune-checkpoint-inhibitor induced pneumonitis.

JAK-signaling cytokines also play a major role in the activation of Tcells, a sub-type of immune cells that is central to many immuneprocesses. Pathological T cell activation is critical in the etiology ofmultiple respiratory diseases. Autoreactive T cells play a role inbronchiolitis obliterans organizing pneumonia (also termed COS). Similarto COS the etiology of lung transplant rejections is linked to anaberrant T cell activation of the recipients T cells by the transplanteddonor lung. Lung transplant rejections may occur early as Primary GraftDysfunction (PGD), organizing pneumonia (OP), acute rejection (AR) orlymphocytic bronchiolitis (LB) or they may occur years after lungtransplantation as Chronic Lung Allograft Dysfunction (CLAD). CLAD waspreviously known as bronchiolitis obliterans (BO) but now is considereda syndrome that can have different pathological manifestations includingBO, restrictive CLAD (rCLAD or RAS) and neutrophilic allograftdysfunction. Chronic lung allograft dysfunction (CLAD) is a majorchallenge in long-term management of lung transplant recipients as itcauses a transplanted lung to progressively lose functionality (Gauthieret al., Curr Transplant Rep., 2016, 3(3), 185-191). CLAD is poorlyresponsive to treatment and therefore, there remains a need foreffective compounds capable of preventing or treating this condition.Several JAK-dependent cytokines such as IFNγ and IL-5 are up-regulatedin CLAD and lung transplant rejection (Berastegui et al, ClinTransplant. 2017, 31, e12898). Moreover, high lung levels of CXCR3chemokines such as CXCL9 and CXCL10 which are downstream ofJAK-dependent IFN signaling, are linked to worse outcomes in lungtransplant patients (Shino et al, PLOS One, 2017, 12 (7), e0180281).Systemic JAK inhibition has been shown to be effective in kidneytransplant rejection (Vicenti et al., American Journal ofTransplantation, 2012, 12, 2446-56). Therefore, JAK inhibitors have thepotential to be effective in treating or preventing lung transplantrejection and CLAD. Similar T cell activation events as described as thebasis for lung transplant rejection also are considered the main driverof lung graft-versus-host disease (GVHD) which can occur posthematopoietic stem cell transplants. Similar to CLAD, lung GVHD is achronic progressive condition with extremely poor outcomes and notreatments are currently approved. A retrospective, multicenter surveystudy of 95 patients with steroid-refractory acute or chronic GVHD whoreceived the systemic JAK inhibitor ruxolitinib as salvage therapydemonstrated complete or partial response to ruxolitinib in the majorityof patients including those with lung GVHD (Zeiser et al, Leukemia,2015, 29, 10, 2062-68). As systemic JAK inhibition is associated withserious adverse events and a small therapeutic index, the need remainsfor an inhaled lung-directed, non-systemic JAK inhibitor to preventand/or treat lung transplant rejection or lung GVHD.

Accordingly, the disclosure further provides a method of treating theadditional respiratory conditions described above in a mammal, themethod comprising administering to the mammal compound 1, or apharmaceutically acceptable salt thereof.

Gastrointestinal Inflammatory Disease

As a JAK inhibitor, compound 1, or a pharmaceutical salt thereof, mayalso be useful to treat gastrointestinal inflammatory diseases thatinclude, but are not limited to, inflammatory bowel disease, ulcerativecolitis (proctosigmoiditis, pancolitis, ulcerative proctitis andleft-sided colitis), Crohn's disease, collagenous colitis, lymphocyticcolitis, Behcet's disease, celiac disease, immune checkpoint inhibitorinduced colitis, ileitis, eosinophilic esophagitis, graft versus hostdisease-related colitis, and infectious colitis. Ulcerative colitis(Reimund et al., J Ciin Immunology, 1996, 16, 144-150), Crohn's disease(Woywodt et al., Eur J Gastroenterology Hepatology, 1999, 11, 267-276),collagenous colitis (Kumawat et al., Mol Immunology, 2013, 55, 355-364),lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis(Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260), graftversus host disease-related colitis (Coghill et al., Blood, 2001, 117,3268-3276), infectious colitis (Stallmach et al., Int J Colorectal Dis,2004, 19, 308-315), Behcet's disease (Zhou et al., Autoimmun Rev, 2012,11, 699-704), celiac disease (de Nitto et al., World J Gastroenterol,2009, 15, 4609-4614), immune checkpoint inhibitor induced colitis (e.g.,CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014,12, 191), PD-1- or PD-L1-inhibitor-induced colitis), and ileitis(Yamamoto et al., Dig Liver Dis, 2008, 40, 253-259) are characterized byelevation of certain pro-inflammatory cytokine levels. As manypro-inflammatory cytokines signal via JAK activation, compoundsdescribed in this application may be able to alleviate the inflammationand provide symptom relief.

Inflammatory Skin Disease

Atopic dermatitis and other inflammatory skin diseases have beenassociated with elevation of proinflammatory cytokines that rely on theJAK-STAT pathway. Therefore compound 1, or a pharmaceutical saltthereof, may be beneficial in a number of dermal inflammatory orpruritic conditions that include, but are not limited to atopicdermatitis, alopecia areata, vitiligo, psoriasis, dermatomyositis,cutaneous T cell lymphoma (Netchiporouk et al., Cell Cycle. 2014; 13,3331-3335) and subtypes (Sezary syndrome, mycosis fungoides, pagetoidreticulosis, granulomatous slack skin, lymphomatoid papulosis,pityriasis lichenoides chronica, pityriasis lichenoides et varioliformisacuta, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ largecell lymphoma, non-mycosis fungoides CD30− cutaneous large T-celllymphoma, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneousT-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma),prurigo nodularis, lichen planus, primary localized cutaneousamyloidosis, bullous pemphigoid, skin manifestations of graft versushost disease, pemphigoid, discoid lupus, granuloma annulare, lichensimplex chronicus, vulvar/scrotal/perianal pruritus, lichen sclerosus,post herpetic neuralgia itch, lichen planopilaris, and foliculitisdecalvans. In particular, atopic dermatitis (Bao et al., JAK-SAT, 2013,2, e24137), alopecia areata (Xing et al., Nat Med, 2014, 20, 1043-1049),vitiligo (Craiglow et al, JAMA Dermatol. 2015, 151, 1110-1112), prurigonodularis (Sonkoly et al., J Allergy Clin Immunol. 2006, 117, 411-417),lichen planus (Welz-Kubiak et al., J Immunol Res. 2015, ID:854747),primary localized cutaneous amyloidosis (Tanaka et al., Br J Dermatol.2009, 161, 1217-1224), bullous pemphigoid (Feliciani et al., Int JImmunopathol Pharmacol. 1999, 12, 55-61), and dermal manifestations ofgraft versus host disease (Okiyama et al., J Invest Dermatol. 2014, 134,992-1000) are characterized by elevation of certain cytokines thatsignal via JAK activation. Accordingly, compound 1, or apharmaceutically acceptable salt thereof, may be able to alleviateassociated dermal inflammation or pruritus driven by these cytokines.

Other Diseases

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat other diseases such as other inflammatory diseases,autoimmune diseases or cancers. Compound 1, or a pharmaceuticallyacceptable salt thereof, may be useful to treat one or more ofarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis,transplant rejection, xerophthalmia, psoriatic arthritis, diabetes,insulin dependent diabetes, motor neurone disease, myelodysplasticsyndrome, pain, sarcopenia, cachexia, septic shock, systemic lupuserythematosus, leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, acute lymphoblastic leukemia, acute myelogenousleukemia, ankylosing spondylitis, myelofibrosis, B-cell lymphoma,hepatocellular carcinoma, Hodgkins disease, breast cancer, Multiplemyeloma, melanoma, non-Hodgkin lymphoma, non-small-cell lung cancer,ovarian clear cell carcinoma, ovary tumor, pancreas tumor, polycythemiavera, Sjoegrens syndrome, soft tissue sarcoma, sarcoma, splenomegaly,T-cell lymphoma, and thalassetnia major.

Combination Therapy

Compound 1 of the disclosure or a pharmaceutically acceptable saltthereof may be used in combination with one or more agents which act bythe same mechanism or by different mechanisms to treat a disease. Thedifferent agents may be administered sequentially or simultaneously, inseparate compositions or in the same composition. Useful classes ofagents for combination therapy include, but are not limited to, a beta 2adrenoceptor agonist, a muscarinic receptor antagonist, a glucocorticoidagonist, a G-protein coupled receptor-44 antagonist, a leukotriene D4antagonist, a muscarinic M3 receptor antagonist, a histamine HT receptorantagonist, an immunoglobulin E antagonist, a PDE 4 inhibitor, an IL-4antagonist, a muscarinic M1 receptor antagonist, a histamine receptorantagonist, an IL-13 antagonist, an IL-5 antagonist, a 5-Lipoxygenaseinhibitor, a beta adrenoceptor agonist, a CCR3 chemokine antagonist, aCFTR stimulator, an immunoglobulin modulator, an interleukin 33 ligandinhibitor, a PDE 3 inhibitor, a phosphoinositide-3 kinase deltainhibitor, a thromboxane A2 antagonist, an elastase inhibitor, a Kittyrosine kinase inhibitor, a leukotriene E4 antagonist, a leukotrieneantagonist, a PGD2 antagonist, a TNF alpha ligand inhibitor, a TNFbinding agent, a complement cascade inhibitor, an eotaxin ligandinhibitor, a glutathione reductase inhibitor, an histamine H4 receptorantagonist, an IL6 antagonist, an IL2 gene stimulator, an immunoglobulingamma Fc receptor IIB modulator, an interferon gamma ligand, aninterleukin 13 ligand inhibitor, an interleukin 17 ligand inhibitor, aL-Selectin antagonist, a leukocyte elastase inhibitor, a leukotriene C4antagonist, a Leukotriene C4 synthase inhibitor, a membrane copper amineoxidase inhibitor, a metalloprotease-12 inhibitor, a metalloprotease-9inhibitor, a mite allergen modulator, a muscarinic receptor modulator, anicotinic acetylcholine receptor agonist, a nuclear factor kappa Binhibitor, a p-Selectin antagonist, a PDE 5 inhibitor, a PDGF receptorantagonist, a phosphoinositide-3 kinase gamma inhibitor, a TLR-7agonist, a TNF antagonist, an Abl tyrosine kinase inhibitor, anacetylcholine receptor antagonist, an acidic mammalian chitinaseinhibitor, an ACTH receptor agonist, an actin polymerization modulator,an adenosine A1 receptor antagonist, an adenylate cyclase stimulator, anadrenoceptor antagonist, an adrenocorticotrophic hormone ligand, analcohol dehydrogenase 5 inhibitor, an alpha 1 antitrypsin stimulator, analpha 1 proteinase inhibitor, an androgen receptor modulator, anangiotensin converting enzyme 2 stimulator, an ANP agonist, a Bcrprotein inhibitor, a beta 1 adrenoceptor antagonist, a beta 2adrenoceptor antagonist, a beta 2 adrenoceptor modulator, a beta amyloidmodulator, a BMP10 gene inhibitor, a BMP15 gene inhibitor, a calciumchannel inhibitor, a cathepsin G inhibitor, a CCL26 gene inhibitor, aCCR3 chemokine modulator, a CCR4 chemokine antagonist, a cell adhesionmolecule inhibitor, a chaperonin stimulator, a chitinase inhibitor, acollagen I antagonist, a complement C3 inhibitor, a CSF-1 antagonist, aCXCR2 chemokine antagonist, a cytokine receptor common beta chainmodulator, a cytotoxic T-lymphocyte protein-4 stimulator, adeoxyribonuclease I stimulator, a deoxyribonuclease stimulator, adipeptidyl peptidase I inhibitor, a DNA gyrase inhibitor, a DPprostanoid receptor modulator, an E-Selectin antagonist, an EGFR familytyrosine kinase receptor inhibitor, an elastin modulator, an EndothelinET-A antagonist, an Endothelin ET-B antagonist, an epoxide hydrolaseinhibitor, a FGF3 receptor antagonist, a Fyn tyrosine kinase inhibitor,a DATA 3 transcription factor inhibitor, a Glucosylceramidase modulator,a Glutamate receptor modulator, a GM-CSF ligand inhibitor, a Guanylatecyclase stimulator, a H+ K+ ATPase inhibitor, an hemoglobin modulator,an Heparin agonist, an Histone deacetylase inhibitor, an Histonedeacetylase-2 stimulator, an HMG CoA reductase inhibitor, an I-kappa Bkinase beta inhibitor, an ICAM1 gene inhibitor, an IL-17 antagonist, anIL-17 receptor modulator, an IL-23 antagonist, an IL-4 receptormodulator, an Immunoglobulin G modulator, an Immunoglobulin G1 agonist,an Immunoglobulin G1 modulator, an Immunoglobulin epsilon Fc receptor IAantagonist, an Immunoglobulin gamma Fc receptor IIB antagonist, anImmunoglobulin kappa modulator, an Insulin sensitizer, an Interferonbeta ligand, an Interleukin 1 like receptor antagonist, an Interleukin18 ligand inhibitor, an Interleukin receptor 17A antagonist, anInterleukin-1 beta ligand inhibitor, an Interleukin-5 ligand inhibitor,an Interleukin-6 ligand inhibitor, a KCNA voltage-gated potassiumchannel-3 inhibitor, a Kit ligand inhibitor, a Laminin-5 agonist, aLeukotriene CysLT1 receptor antagonist, a Leukotriene CysLT2 receptorantagonist, a LOXL2 gene inhibitor, a Lyn tyrosine kinase inhibitor, aMARCKS protein inhibitor, a MDR associated protein 4 inhibitor, aMetalloprotease-2 modulator, a Metalloprotease-9 modulator, aMineralocorticoid receptor antagonist, a Muscarinic M2 receptorantagonist, a Muscarinic M4 receptor antagonist, a Muscarinic M5receptor antagonist, a Natriuretic peptide receptor A agonist, a Naturalkiller cell receptor modulator, a Nicotinic ACh receptor alpha 7 subunitstimulator, a NK cell receptor modulator, a Nuclear factor kappa Bmodulator, an opioid growth factor receptor agonist, a P-Glycoproteininhibitor, a P2X3 purinoceptor antagonist, a p38 MAP kinase inhibitor, aPeptidase 1 modulator, a phospholipase A2 inhibitor, a phospholipase Cinhibitor, a plasminogen activator inhibitor 1 inhibitor, a plateletactivating factor receptor antagonist, a PPAR gamma agonist, aprostacyclin agonist, a protein tyrosine kinase inhibitor, a SH2 domaininositol phosphatase I stimulator, a signal transduction inhibitor, asodium channel inhibitor, a STAT-3 modulator, a Stem cell antigen-1inhibitor, a superoxide dismutase modulator, a T cell surfaceglycoprotein CD28 inhibitor, a T-cell surface glycoprotein CD8inhibitor, a TGF beta agonist, a TGF beta antagonist, a thromboxanesynthetase inhibitor, a thymic stromal lymphoprotein ligand inhibitor, athymosin agonist, a thymosin beta 4 ligand, a TLR-8 agonist, a TLR-9agonist, a TLR9 gene stimulator, a Topoisomerase IV inhibitor, aTroponin I fast skeletal muscle stimulator, a Troponin T fast skeletalmuscle stimulator, a Type I IL-1 receptor antagonist, a Type II TNFreceptor modulator, an ion channel modulator, a uteroglobin stimulator,and a VIP agonist.

Specific agents that may be used in combination with the present JAKinhibitor compound 1 include, but are not limited to rosiptor acetate,umeclidinium bromide, secukinumab, metenkefalin acetate, tridecactideacetate, fluticasone propionate, alpha-cyclodextrin-stabilizedsulforaphane, tezepelumab, mometasone furoate, BI-1467335, dupilumab,aclidinium, formoterol, AZD-1419, HI-1640V, rivipansel, CMP-001,mannitol, ANB-020, omalizumab, tregalizumab, Mitizax, benralizumab,golimumab, roflumilast, imatinib, REGN-3500, masitinib, apremilast,RPL-554, Actimmune, adalimumab, rupatadine, parogrelil, MK-1029,beclometasone dipropionate, formoterol fumarate, mogamulizumab,seratrodast, LCB-4144, nemiralisib, CK-2127107, fevipiprant, danirixin,bosentan, abatacept, EC-18, duvelisib, dociparstat, ciprofloxacin,salbutamol HFA, erdosteine, PrEP-001, nedocromil, CDX-0158, salbutamol,enobosarm, R-TPR-022, lenzilumab, fluticasone furoate, vilanteroltrifenatate, fluticasone propionate, salmeterol, PT-007, PRS-060,remestemcel-L, citrulline, RPC-4046, nitric oxide, DS-102, gerilimzumab,Actair, fluticasone furoate, umeclidinium, vilanterol, AG-NPP709,Gamunex, infliximab, Ampion, acumapimod, canakinumab, INS-1007, CYP-001,sirukumab, fluticasone propionate, mepolizumab, pitavastatin,solithromycin, etanercept, ivacaflor, anakinra, MPC-300-IV,glycopyrronium bromide, aclidinium bromide, FP-025, risankizumab,glycopyrronium, formoterol fumarate, Adipocell, YPL-001, tiotropiumbromide, glycopyrronium bromide, indacaterol maleate, andecaliximab,olodaterol, esotneprazole, dust mite vaccine, mugwort pollen allergenvaccine, vamorolone, gefapixant, revefenacin, gefitinib, ReJoin,tipelukast, bedoradrine, SCM-CGH, SHP-652, RNS-60, brodalumab,BIO-11006, umeclidinium bromide, vilanterol trifenatate, ipratropiumbromide, tralokinumab, PUR-1800, VX-561, VX-371, olopatadine,tulobuterol, formoterol fumarate, triamcinolone acetonide, reslizumab,salmeterol xinafoate, fluticasone propionate, beclometasonedipropionate, formoterol fumarate, tiotropium bromide, ligelizumab,RUTI, bertilimumab, omalizumab, glycopyrronium bromide, SENS-111,beclomethasone dipropionate, CHF-5992, LT-4001, indacaterol,glycopyrronium bromide, mometasone furoate, fexofenadine, glycopyrroniumbromide, azithromycin, AZD-7594, formoterol, CHF-6001, batefenterol,OATD-01, olodaterol, CJM-112, rosiglitazone, salmeterol, setipiprant,inhaled interferon beta, AZD-8871, plecanatide, fluticasone, salmeterol,eicosapentaenoic acid monoglycerides, lebrikizutnab, RG-6149, QBKPN,Mometasone, indacaterol, AZD-9898, sodium pyruvate, zileuton, CG-201,imidafenacin, CNTO-6785, CUBS-03, mometasone, RGN-137, procaterol,formoterol, CCI-15106, POL-6014, indacaterol, beclomethasone, MV-130,GC-1112, Allergovac depot, MEDI-3506, QBW-251, ZPL-389, udenafil,GSK-3772847, levocetirizine, AXP-1275, ADC-3680, timapiprant,abediterol, AZD-7594, ipratropium bromide, salbutamol sulfate, tadekinigalfa, ACT-774312, dornase alfa, iloprost, batefenterol, fluticasonefuroate, alicaforsen, ciclesonide, emeramide, arformoterol, SB-010,Ozagrel, BTT-1023, Dectrekumab, levalbuterol, pranlukast, hyaluronicacid, GSK-2292767, Formoterol, NOV-14, Lucinactant, salbutamol,prednisolone, ebastine, dexamethasone cipecilate, GSK-2586881,BI-443651, GSK-2256294, VR-179, VR-096, hdm-ASIT+, budesonide,GSK-2245035, VTX-1463, Emedastine, dexpramipexole, levalbuterol, N-6022,dexamethasone sodium phosphate, PIN-201104, OPK-0018, TEV-48107,suplatast, BI-1060469, Gemilukast, interferon gamma, dalazatide,bilastine, fluticasone propionate, salmeterol xinafoate, RP-3128,bencycloquidium bromide, reslizumab, PBF-680, CRTH2 antagonist,Praniukast, salmeterol xinafoate, fluticasone propionate, tiotropiumbromide monohydrate, masilukast, RG-7990, Doxofylline, abediterol,glycopyrronium bromide, TEV-46017, ASM-024, fluticasone propionate,glycopyrronium bromide, salmeterol xinafoate, salbutamol, TA-270,Flunisolide, sodium chromoglycate, Epsi-gam, ZPL-521, salbutamol,aviptadil, TRN-157, Zafirlukast, Stempeucel, pemirolast sodium, nadolol,fluticasone propionate+salmeterol xinafoate, RV-1729, salbutamolsulfate, carbon dioxide+perfluorooctyl bromide, APL-1,dectrekumab+VAK-694, lysine acetylsalicylate, zileuton, TR-4, humanallogenic adipose-derived mesenchymal progenitor cell therapy,MEDI-9314, PL-3994, HMP-301, TD-5471, NKTT-120, pemirolast,beclomethasone dipropionate, trantinterol, monosodium alpha luminol,IMD-1041, AM-211, TBS-5, ARRY-502, seratrodast, recombinant midismase,ASM-8, deflazacort, bambuterol, RBx-10017609, ipratropium+fenoterol,fluticasone+formoterol, epinastine, WIN-901X, VALERGEN-DS,OligoG-COPD-5/20, tulobuterol, oxis Turbuhaler, DSP-3025, ASM-024,mizolastine, budesonide+salmeterol, LH-011, AXP-E, histamine humanimmunoglobulin, YHD-001, theophylline, ambroxol+erdosteine, ramatroban,montelukast, pranlukast, AG-1321001, tulobuterol,ipratropium+salbutamol, tranilast, methylprednisolone suleptanate,colforsin daropate, repirinast, and doxofylline.

Also provided, herein, is a pharmaceutical composition comprisingcompound 1, or a pharmaceutically acceptable salt thereof and one ormore other therapeutic agents. The therapeutic agent may be selectedfrom the class of agents specified above and from the list of specificagent described above. In some embodiments, the pharmaceuticalcomposition is suitable for delivery to the lungs. In some embodiments,the pharmaceutical composition is suitable for inhaled or nebulizedadministration. In some embodiments, the pharmaceutical composition is adry powder or a liquid composition.

Further, in a method aspect, the invention provides a method of treatinga disease or disorder in a mammal comprising administering to the mammalcompound 1 or a pharmaceutically acceptable salt thereof and one or moreother therapeutic agents.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, or the agents may be provided inseparate compositions that are administered simultaneously or atseparate times, by the same or by different routes of administration.Such compositions can be packaged separately or may be packaged togetheras a kit. The two or more therapeutic agents in the kit may beadministered by the same route of administration or by different routesof administration.

The compound of the invention has been demonstrated to be a potentinhibitor of the JAK1, JAK2, JAK3, and TYK2 enzymes in enzyme bindingassays, to have potent functional activity without cytotoxicity incellular assays, and to exert the pharmacodynamic effects of JAKinhibition in preclinical models, as described in the followingexamples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   ACN=acetonitrile    -   DCC=dicyclohexylcarbodiimide    -   DATA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   EtOAc=ethyl acetate    -   HATU=N,N,N′N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   LDA=lithium diisopropylamide    -   min=minute(s)    -   MTBE=methyl tert-butyl ether    -   NBS=N-bromosuccinimide    -   RT=room temperature    -   THF=tetrahydrofuran    -   bis(pinacolato)diboron=4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl]    -   Pd(dppf)Cl₂—CH₂Cl₂=dichloro(1,1′-bis(diphenylphosphino)-ferrocene)-dipalladium(1.1)        complex with dichloromethane

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions

Column: C18, 5 μm 21.2×150 mm or C18, 5 μm 21×250 or

-   -   C14, 5 μm 21×150 mm        Column temperature: Room Temperature        Flow rate: 20.0 mL/min        Mobile Phases: A=Water+0.05% TFA    -   B=ACN+0.05% TFA,        Injection volume: (100-1500 μL)        Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Analytic HPLC Conditions

Method A

Column: Agilent Zorbax Bonus-RP C18, 150×4.60 nm, 3.5 micron

Column temperature: 40° C.

Flow rate: 1.5 mL/min

Injection volume: 5 μL

Sample preparation: Dissolve in 1:1 ACN:1 M HCl

Mobile Phases: A=Water: TFA (99.95:0.05)

-   -   B=ACN:TFA (99.95:0.05)        Detector wavelength: 254 nm and 214 nm        Gradient: 26 min total (time (min)/% B): 0/5, 18/90, 22/90,        22.5/90, 26/5        Method B        Column: Agilent Poroshell 120 Bonus-RP, 4.6×150 mm, 2.7 μm.        Column temperature: 30° C.        Flow rate: 1.5 mL/min        Injection volume: 10 μL        Mobile Phases: A=ACN:Water:TFA (2:98:0.1)    -   B=ACN:Water:TFA. (90:10:0.1)        Sample preparation: Dissolve in Mobile phase B        Detector wavelength: 254 nm and 214 nm        Gradient: 60 mm total (time (min)/% B): 0/0, 50/100, 55/100,        55.1/0, 60/0

Preparation 1: 1-benzyl-4-imino-1,4-dihydropyridin-3-amine

A mixture of pyridine-3,4-diamine (445 g, 4.078 mol) and ACN (11.0 L)was stirred for 80 min from 25° C. to 15° C. Benzyl bromide (485 mL,4.078 mol) was added over 20 min and the reaction mixture was stirred at20° C. overnight. The reaction mixture was cooled to 10° C. andfiltered. To the reactor was added ACN (3 L), which was cooled to 10° C.The cake was washed with the reactor rinse and washed again with ACN (3L) warmed to 25° C. The solid was dried on the filter for 24 h undernitrogen, at 55° C. under vacuum for 2 h and then at RT overnight andfor 4 d to provide the HBr salt of the title compound (1102.2 g, 3.934mol, 96% yield). HPLC Method A Retention time 4.12 min.

Preparation 2:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

(a) 5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

A solution of 6-bromo-1H-indazole-3-carbaldehyde (550 g, 2.444 mol),1-benzyl-4-imino-1,4-dihydropyridin-3-amine HBr (721 g, 2.333 mol) andDMAc (2.65 L) was stirred for 60 min and sodium bisulfite (257 g, 2.468mol) was added. The reaction mixture was heated to 135° C. and held for3 h, and allowed to cool to 20° C. and held at 20° C. overnight.Acetonitrile (8 L) was added and the reaction mixture was stirred for 4h at 15° C. The slurry was filtered on a pressure filter at mediumfiltration rate. To the reactor was added ACN (1 L) The cake was washedwith the ACN reactor wash and dried under nitrogen overnight and thenunder vacuum at 50° C. for 24 h to provide the HBr salt of the titlecompound (1264 g, 2.444 mol, 100% yield, 94% purity) as a dense wetbeige/brown solid. HPLC Method A Retention time 8.77 min.

A mixture of the product of the previous step (1264 g, 2.444 mol), MeTHF(6 L) and water (2.75 L) was heated to 65° C. and sodium hydroxide 50 wt% (254 g, 3.177 mol) was added over 5 min and the reaction mixture wasstirred at 65° C. for 1 h, cooled to RT, then to 5° C. and held for 2 h.The slurry was filtered and the reactor and cake were washed with MeTHF(1 L). The resulting beige to yellow solid was dried on the filter undernitrogen for 3 d to provide the title compound (475 g, 1.175 mmol, 48%yield) as a beige/yellow solid. The mother liquor (about 8 L) wasconcentrated to about 2 L, whereupon solids began to crash out. Theslurry was heated to 50° C., held for 2 h, cooled to 5° C. over 2 h,stirred overnight, and filtered. The cake was washed with MeTHF (100 mL)and dried overnight under vacuum at 40° C. to provide additional titlecompound (140 g, 0.346 mol, 14% yield).

A mixture of the total product of the previous step, combined with theproduct of a second batch at the same scale (1500 g, 3.710 mol) andMeTHF (4 L) was stirred at 20° C. for 2 h and filtered. The reactor andcake were washed with MeTHF (1.5 L). The resulting beige to yellow solidwas dried under nitrogen for 3 d to provide the title compound as abeige yellow solid (1325 g, 3.184 mol, 86% yield (overall 68% yield),97% purity). HPLC Method A Retention time 8.77 min

Preparation 3:5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

To a 15 L flask was added5-benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine (440 g,1.088 mol) followed by MeTHF (4.5 L), methanol (2.25 L) and water (1.125L). The slurry was cooled to 20° C., stirred for 1 h, and NaBH₄ (247 g,6.530 mol) was added. The reaction mixture was stirred at 25° C. for 18h. Water (1.125 L) was added followed by 20 wt %. sodium chloridesolution (1.125 L) and the mixture was stirred for 30 min and the layersallowed to separate. The aqueous layer was drained. A premixed solutionof NaOH (522 g) and water (5 L) was added and the reaction mixture wasstirred for 60 min; the layers were allowed to separate and the aqueouslayer was drained. Two additional batches at the same scale wereprepared.

The organic layer from one batch was concentrated under reduced pressurein a 15 L jacketed reactor with the jacket set at 50° C., internaltemperature 20° C. The additional batches were added to the reactor andconcentrated one at a time resulting in a slurry about 6 L in volume.The slurry was heated to 50° C., IPAc (6 L) was added and the mixturewas held at 60° C. for 1.5 h, cooled to 20° C. for 10 h, heated to 60°C. for 50 h, cooled to 20° C. in 5 h, then cooled to 5° C. and held for3 h. The mixture was filtered and the reactor and cake was washed with apremixed solution of IPAc (1 L) and MeTHF (1 L), precooled to 5° C. Thesolids were dried under nitrogen on the filter at 40° C. for 3 d toprovide the title compound (1059 g, 2.589 mol, 79% yield) as anoff-white solid. The material was further dried in a vacuum oven at50-60° C. for 8 h and at 27° C. for 2 d to provide the title compound(1043 g, 2.526 mol, 77% yield, 99% purity). HPLC Method A Retention time6.73 min.

Preparation 4: (4-(Benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate,potassium

(a)2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of 1-(benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (520 g, 1682mmol) and dioxane (5193 mL) was purged with nitrogen and thenbis(pinacolato)diboron (641 g, 2523 mmol) was added followed bypotassium acetate (495 g, 5046 mmol). The reaction mixture was purgedwith nitrogen; Pd(dppf)Cl₂ (41.2 g, 50.5 mmol) was added; the reactionmixture was purged with nitrogen, heated at 103° C. under nitrogen for 5h; and cooled to RT. The reaction mixture was concentrated by vacuumdistillation and partitioned between ethyl acetate (5204 mL) and water(5212 mL). The reaction mixture was filtered through Celite; the organiclayer was washed with brine (2606 mL) followed by solvent removal byvacuum distillation to provide crude product as a thick black oil (˜800g).

The crude product was dissolved in DCM (1289 mL) and purified by silicagel chromatography (2627 g silica preslurried in hexane, eluted with 20%ethyl acetate in hexanes (10.35 L)). Solvent was removed by vacuumdistillation to yield a light yellow oil (600 g). HPLC Method BRetention time 33.74 min.

(b) (4-(benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate, potassium

The product of the previous step (200 g, 561 mmol) was mixed withacetone (1011 mL) until complete dissolution and methanol (999 mL) wasadded followed by 3 M potassium hydrogen difluoride (307 g, 3930 mmol)dissolved in water (1310 mL). The reaction mixture was stirred for 3.5h. Most of the organic solvent was removed by vacuum distillation. Water(759 mL) was added and the resulting thick slurry was stirred for 30 minand filtered. The cake was washed with water (506 mL) and the solidswere dried on the filter for 30 min. The solids were slurried in acetone(1237 mL) and stirred for 1 h. The resulting slurry was filtered and thesolids washed with acetone (247 mL). The acetone solution wasconcentrated by vacuum distillation, and a constant volume (2 L) wasmaintained by slow addition of toluene (2983 mL) until all acetone andwater had been distilled. The toluene solution was distilled to a thickyellow slurry by rotary evaporation, during which time the productsprecipitated as white solids. An additional portion of toluene (477 mL)was added to the mixture and stirred for 1 h. The mixture was thenfiltered and rinsed with toluene (179 mL) and dried under vacuum at 50°C. for 24 h to provide the title compound (104 g, 310 mmol, 55% yield)as a free-flowing, fluffy, slightly off-white solid. HPLC Method BRetention time 27.71 min.

Preparation 5:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

(a)5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

A mixture of bis(pinacolato)diboron (250 g, 984 mmol) and IPA (1.88 vasstirred to dissolution and then a solution of potassium hydrogendifluoride (538 g, 6.891 mol) in water (2.31 L) was added portion-wiseover 10 min. The reaction mixture was stirred for 1 h and filtered. Thegel-like solids were slurried with water (1.33 L) until the mixtureformed a clear hydrogel and then for another 45 min. The resultingsolids/gel were filtered, then reslurried in acetone (1.08 L), filtered,air dried on the filter for 30 min and dried overnight to provide afluffy white solid (196.7 g).

To a 5 L flask was added5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(135 g, 331 mmol),(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-trifluoroborate, potassium (133g, 397 mmol), and the white solid product of the previous step (40.5 g)followed by MeTHF (1.23 L) and MeOH (1.75 L). The resulting slurry wasdegassed three times with nitrogen. To the slurry was added a degassedsolution of cesium carbonate (431 g, 1.323 mol) in water (1.35 L). Theslurry was degassed twice, Pd (amphos)₂Cl₂ (11.71 g, 16.53 mmol) wasadded, the slurry was again degassed twice and the reaction mixture wasstirred at 67° C. overnight and cooled to 20° C. The layers wereseparated and back extracted with MeTHF (550 mL). The organic layerswere combined and concentrated by rotary evaporation until solidsprecipitated. MeTHF (700 mL) was added and the reaction mixture wasstirred at 65° C. The layers were separated and the aqueous phase backextracted with MeTHF (135 mL). The organic phases were combined andconcentrated to about 300 mL resulting in a thick orange slurry. To theslurry was added MeOH (270 mL) followed by 1M HCl (1.325 L) at 20° C.with rapid stirring. The reaction mixture was stirred for 5 min andwater (1 L) was added and the resulting slurry was stirred for 1 h. Thesolids were filtered, washed with water (150 mL), dried on the filterfor 10 min and at 45° C. under nitrogen for 16 h to provide the 2 HClsalt of the title compound (221.1 g, 351 mmol, 92.2% purity) as a lightyellow solid. HPLC Method B retention time 23.41 mm.

Preparation 6:5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a 1 L flask was added5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine,2 HCl (40 g, 63.4 mmol) as a slurry in ethanol 348 mL) and 1.25 M HCl inMeOH (101 mL) and water (17.14 mL). The reaction mixture was degassedwith nitrogen for 5 min and 10 wt % Pd/C, 50 wt % H₂O (4.05 g, 1.903mmol) was added. The reactor was sealed, purged with H₂ pressurized to1-2 psi. warmed to 50° C., and the reaction mixture was stirredovernight and filtered through Celite. The reactor and filter werewashed with methanol (100 mL).

The filtered solution was combined with the product of a second batch atthe 98 mmol scale and concentrated to 390 g. EtOAc (2.04 L) was addedslowly with stirring and then the solution was cooled to 5° C. withstirring. Solids were filtered, washed with EtOAc (510 mL), and driedovernight at 45° C. under nitrogen to provide the 2 HCl salt of thetitle compound (58 g, 80% yield) as an off-white solid. HPLC Method Bretention time 12.83 min.

Example 1: Crystalline Hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a 3 L flask was added NMP (239 mL) and5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,2 HCl (74.5 g, 165 mmol) with stirring followed by NMP (74 mL). Aceticacid (31.3 mL) was added and the reaction mixture was warmed to 55° C.for 10 min and then cooled to 25° C. 1-methylpiperidin-4-one (61.0 mL,496 mmol) was added in a single portion and the reaction mixture wasstirred at 25° C. for 30 min and cooled to 15° C. Sodiumtriacetoxyborohydride (98 g, 463 mmol) was added and the external jacketwas set to 20° C. after 5 min. After 3 h, ammonium hydroxide (365 mL,5790 mmol) was added dropwise over 45 min maintaining the temperaturebelow 25° C. The reaction mixture was stirred for 1.5 h at 20° C.,forming an off-white slurry. Methanol (709 mL) was added and thereaction mixture was stirred slowly overnight at 55° C. Water (1.19 L)was added over 30 min at 55° C. and the mixture was cooled to 10° C.,stirred for 2 h, and filtered. The cake was washed with 1:1 MeOH:water(334 mL), dried on the filter for 20 min and at 45° C. under vacuum withnitrogen bleed to provide yellow solids (87 g).

To the solids was added 5% water/acetone (1.5 L) at 55° C. with slowstirring and the reaction mixture was heated at 55° C. for 6 h, cooledto 10° C., filtered, and washed with 5% water/acetone (450 mL). Thesolids were dried overnight at 50° C. under vacuum with nitrogen bleed,equilibrated in air for 20 h, dried in the vacuum oven for 48 h andequilibrated with air to provide the title compound (71.3 g, 91% yield)as a free flowing pale yellow solid. HPLC Method B Retention time 12.29min.

Example 2: Powder X-Ray Diffraction

The powder X-ray diffraction (PXRD) pattern of the product of Example 1was obtained with a Bruker D8-Advance X-ray diffractometer using Cu-Kαradiation (λ=1.54051 Å) with output voltage of 45 kV and current of 40mA. The instrument was operated in Bragg-Brentano geometry withincident, divergence, and scattering slits set to maximize the intensityat the sample. For measurement, a small amount of powder (5-25 mg) wasgently pressed onto a sample holder to form a smooth surface andsubjected to X-ray exposure. The samples were scanned in 2θ-2θ mode from2° to 40° in 2θ with a step size of 0.02° and a scan speed of 0.30°seconds per step. The data acquisition was controlled by BrukerDiffracSuite measurement software and analyzed by Jade software (version7.5.1). The instrument was calibrated with a corundum standard, within±0.02° two-theta angle. Observed PXRD two-theta peak positions andd-spacings are shown in Table 1.

TABLE 1 PXRD Data for the Crystalline Hydrate 2-Theta d (Å) Area A %6.20 14.24 81639 45.70 9.58 9.22 178629 100.00 10.34 8.55 30022 16.8010.65 8.30 12801 7.20 11.54 7.66 27220 15.20 12.77 6.93 27705 15.5013.01 6.80 48785 27.30 13.39 6.61 9261 5.20 16.94 5.23 40031 22.40 17.535.05 83718 46.90 18.67 4.75 9542 5.30 19.28 4.60 152922 85.60 20.02 4.4322391 12.50 20.61 4.31 30308 17.00 21.51 4.13 92875 52.00 22.10 4.0237495 21.00 22.79 3.90 13802 7.70 23.22 3.83 12117 6.80 25.16 3.54 137927.70 28.80 3.10 14487 8.10 29.62 3.01 14810 8.30 30.20 2.96 9709 5.40

BIOLOGICAL ASSAYS

5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(compound 1) has been characterized in the following biological assays.

Assay 1: Biochemical JAK Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAR enzymes and a GFP-tagged STALL peptide substrate were obtained fromLife Technologies.

The serially diluted compound was pre-incubated with each of the fourJAK enzymes and the substrate in white 384-well microplates (Corning) atambient temperature for 1 h. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for 1 h before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC₅₀)and subsequently converted to pK_(i) (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

The compound of the invention exhibited the following enzyme potency.

TABLE 2 JAK 1 JAK 2 JAK 3 Tyk2 pK_(i) pK_(i) pK_(i) pK_(i) 10.2 10.8 9.79.8

Assay 2: Cellular JAK Potency Assay: Inhibition of IL-13

The AlphaScreen JAKI cellular potency assay was carried out by measuringinterleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation inBEAS-2B human lung epithelial cells (ATCC). The anti-STAT6 antibody(Cell Signaling Technologies) was conjugated to AlphaScreen acceptorbeads (Perkin Elmer), while the anti-pSTAT6 (pTyr641) antibody (CellSignaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin(Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO₂ humidified incubator in50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 7,500 cells/well density in whitepoly-D-lysine-coated 384-well plates (Corning) with 25 μL medium, andwere allowed to adhere overnight in the incubator. On day 2 of theassay, the medium was removed and replaced with 12 μL of assay bufferHank's Balanced Salt Solution/HBSS, 25 mM HEPES, and 1 mg/ml bovineserum albumin/BSA) containing dose-responses of test compounds. Thecompound was serially diluted in DMSO and then diluted another 1000-foldin media to bring the final DMSO concentration to 0.1%. Cells wereincubated with test compounds at 37° C. for 1 h, and followed by theaddition of 12 μl of pre-warmed IL-13 (80 ng/mL in assay buffer) forstimulation. After incubating at 37° C. for 30 min, the assay buffer(containing compound and IL-13) was removed, and 10 μL of cell lysisbuffer (25 mM HEPES, 0.1% SDS, 1% NP-40, 5 mM MgCl₂, 1.3 mM EDTA, 1 mMEGTA, and supplement with Complete Ultra mini protease inhibitors andPhosSTOP from Roche Diagnostics). The plates were shaken at ambienttemperature for 30 min before the addition of detection reagents. Amixture of biotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beadswas added first and incubated at ambient temperature for 2 h, followedby the addition of streptavidin conjugated donor beads (Perkin Elmer).After a minimum of 2 h incubation, the assay plates were read on theEnVision plate reader. AlphaScreen luminescence signals were recordedand utilized to calculate the percent inhibition values based on DMSOand background controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software. Results may alsobe expressed as the negative logarithm of the IC₅₀ value, pIC₅₀. Thecompound of the invention exhibited a pIC₅₀ value of 8.2 in this assay.

Assay 3: Cellular JAK Potency Assay: Inhibition of IL-2/Anti-CD3Stimulated IFNγ in Human PBMCs

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated interferon gamma (INFγ) was measured in humanperipheral blood mononuclear cells (PBMCs) isolated from human wholeblood (Stanford Blood Center). Because IL-2 signals through JAK, thisassay provides a measure of JAK cellular potency.

(1) Human peripheral blood mononuclear cells (PBMC) were isolated fromhuman whole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI: (LifeTechnologies) supplemented with 10% Heat Inactivated Fetal Bovine Serum(FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES(Life Technologies) and 1× Pen/Strep (Life Technologies). Cells wereseeded at 200,000 cells/well in media (50 μL) and cultured for 1 h.Compounds were serially diluted in DMSO and then diluted another500-fold (to a 2× final assay concentration) in media. Test compounds(100 μL/well) were added to cells, and incubated at 37° C., 5% CO₂ for 1h, followed by the addition of IL-2 (R&D Systems; final concentration100 ng/mL) and anti-CD3 (BD Biosciences; final concentration 1 μg/mL) inpre-warmed assay media (50 μL) for 24 h.

(2) After cytokine stimulation, cells were centrifuged at 500 g for 5min and supernatants removed and frozen at −80° C. To determine theinhibitory potency of the test compound in response to IL-2/anti-CD3,supernatant IFNγ concentrations were measured via. ELBA (R&D Systems).IC₅₀ values were determined from analysis of the inhibition curves ofconcentration of IFNγ vs compound concentration. Data are expressed aspIC₅₀ (negative decadic logarithm IC₅₀) values. The compound of theinvention exhibited a pIC₅₀ value of about 7.3 in this assay.

Assay 4: Cellular JAK Potency Assay: Inhibition of IL-2 StimulatedpSTAT5 in CD4+ T Cells

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated STAT5 phosphorylation was measured inCD4-positive (CD4+) T cells in human peripheral blood mononuclear cells(PBMCs) isolated from human whole blood (Stanford Blood Center) usingflow cytometry. Because IL-2 signals through JAK, this assay provides ameasure of JAK cellular potency.

CD4+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD4 antibody (Clone RPA-T4, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT5 antibody (pY694, Clone 47, BD Biosciences)was used to detect STAT5 phosphorylation.

(1) The protocol of Assay 3 paragraph (1) was followed with theexception that the cytokine stimulation with anti-CD3 was performed for30 min instead of 24 h.

(2) After cytokine stimulation, cells were fixed with pre warmed fixsolution (200 μL; BD Biosciences) for 10 min at 37° C., 5% CO₂, washedtwice with DPBS buffer (1 mL, Life Technologies), and resuspended in icecold Perm Buffer III (1000 μL, BD Biosciences) for 30 min at 4° C. Cellswere washed twice with 2. % FBS in DPBS (FACS buffer), and thenresuspended in FACS buffer (100 μL) containing anti-CD4 PE (1:50dilution) and anti-CD3 anti-CD3Alexa Fluor 647 (1:5 dilution) for 60 minat room temperature in the dark. After incubation, cells were washedtwice in FACS buffer before being analyzed using a LSRII flow cytometer(BD Biosciences). To determine the inhibitory potency of test compoundsin response to IL-2/anti-CD3, the median fluorescent intensity (MFI) ofpSTAT5 was measured in CD4+ T cells. IC₅₀ values were determined fromanalysis of the inhibition curves of MFI vs compound concentration. Dataare expressed as pIC₅₀ (negative decadic logarithm IC₅₀) values. Thecompound of the invention exhibited a pIC₅₀ value of about 7.7 in thisassay.

Assay 5: Cellular JAK Potency Assay: Inhibition of IL-4 StimulatedpSTAT6 in CD3+ T Cells

The potency of the test compound for inhibition of interleukin-4 (IL-4)stimulated STAT6 phosphorylation was measured in CD3-positive (CD3+) Tcells in human peripheral blood mononuclear cells (PBMCs) isolated fromhuman whole blood (Stanford Blood Center) using flow cytometry. BecauseIL-4 signals through JAK, this assay provides a measure of JAK cellularpotency.

CD3+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD3 antibody (Clone UCHT1, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT6 antibody (pY641, Clone 18/P, BD Biosciences)was used to detect STAT6 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors as in Assays 3 and 4. Cells were seeded at250,000 cells/well in media (200 μL), cultured for 1 h and thenresuspended in assay media (50 μL) supplemented with 0.1% bovine serumalbumin (Sigma), 2 mM Glutamax, 25 mM HEPES and 1× Penstrep) containingvarious concentrations of test compounds. Compounds were seriallydiluted in DMSO and then diluted another 500-fold (to a 2× final assayconcentration) in assay media. Test compounds (50 μL) were incubatedwith cells at 37° C., 5% CO₂ for 1 h, followed by the addition of IL-4(50 μL) (R&D Systems; final concentration 20 ng/mL) in pre-warmed assaymedia for 30 min. After cytokine stimulation, cells were fixed withpre-warmed fix solution (100 μL) (BD Biosciences) for 10 min at 37° C.5% CO₂, washed twice with FACS buffer (1 mL) (2% FBS in DPBS), andresuspended in ice cold Perm Buffer III (1000 μL) (BD Biosciences) for30 min at 4° C. Cells were washed twice with FACS buffer, and thenresuspended in FACS buffer (100 μL) containing anti-CD3 PE (1:50dilution) and anti-pSTAT6 Alexa Fluor 647 (1:5 dilution) for 60 min atroom temperature in the dark. After incubation, cells were washed twicein FACS buffer before being analyzed using a LSRII flow cytometer (BDBiosciences).

To determine the inhibitory potency of the test compound in response toIL-4, the median fluorescent intensity (MFI) of pSTAT6 was measured inCD3+ T cells. IC₅₀ values were determined from analysis of theinhibition curves of Mil vs compound concentration. Data are expressedas pIC₅₀ (negative decadic logarithm IC₅₀). The compound of theinvention exhibited a pIC₅₀ value of 8.1 in this assay.

Assay 6: Cellular JAK Potency Assay: Inhibition of IL-6 StimulatedpSTAT3 in CD3+ T Cells

A protocol analogous to that of Assay 5 was used to determine thepotency of the test compound for inhibition of interleuken-6 (IL-6)stimulated STAT3 phosphorylation. An Alexa. Fluor 647 conjugatedanti-pSTAT3 antibody (pY705, Clone 4/P, BD Biosciences) was used todetect STAT3 phosphorylation.

The compound of the invention exhibited a pIC₅₀ value of 7.4 in thisassay.

Assay 7: Cellular JAK Potency Assay: Inhibition of IFNγ-Induced pSTAT1

The potency of the test compound for inhibition of interferon gamma(IFNγ) stimulated STAT1 phosphorylation was measured in CD14-positive(CD14+) monocytes derived from human whole blood (Stanford Blood Center)using flow cytometry. Because IFNγ signals through JAK, this assayprovides a measure of JAK cellular potency.

Monocytes were identified using a fluorescein isothiocyanate (FITC)conjugated anti-CD14 antibody (Clone RM052, Beckman Coulter), and anAlexa Fluor 647 conjugated anti-pSTAT1 antibody (pY701, Clone 4a, BDBiosciences) was used to detect STAT1 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPM (LifeTechnologies) supplemented with 10% Fetal Bovine Serum (FBS, LifeTechnologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (LifeTechnologies) and 1× Pen/Strep (Life Technologies). Cells were seeded at250,000 cells/well in media (200 μL), cultured for 2 h and resuspendedin assay media (50 μL) (RPMI supplemented with 0.1% bovine serum albumin(Sigma), 2 mM Glutamax, 25 mM HEPES and 1× Penstrep) containing variousconcentrations of test compounds. The compound was serially diluted inDMSO and then diluted another 1000-fold in media to bring the final DMSOconcentration to 0.1%. Test compound dilutions were incubated with cellsat 37° C., 5% CO₂ for 1 h, followed by the addition of pre-warmed IFNγ(R&D Systems) in media (50 μL) at a final concentration of 0.6 ng/mL for30 min. After cytokine stimulation, cells were fixed with pre-warmed fixsolution (100 μL) (BD Biosciences) for 10 min at 37° C., 5% CO₂, washedtwice with FACS buffer (1 mL) (1% BSA in PBS), resuspended in 1:10anti-CD14 FITC:FACS buffer (100 μL), and incubated at 4° C. for 15 min.Cells were washed once, and then resuspended in ice cold Perm Buffer III(BD Biosciences) (100 μL) for 30 min at 4° C. Cells were washed twicewith FACS buffer, and then resuspended in 1:10 anti-pSTAT1 Alexa. Fluor647:FACS buffer (100 μL) for 30 min at RT in the dark, washed twice inFACS buffer, and analyzed using a LSRII flow cytometer (BD Biosciences).

To determine the inhibitory potency of the test compound, the medianfluorescent intensity (MFI) of pSTAT1 was measured in CD14+ monocytes.IC₅₀ values were determined from analysis of the inhibition curves ofMFI vs compound concentration. Data are expressed as pIC₅₀ (negativedecadic logarithm IC₅₀) values The compound of the invention exhibited apIC₅₀ value of about 7.5 in this assay.

Assay 8: Ocular Pharmacokinetics in Rabbit Eyes

The objective of this assay was to determine the pharmacokinetics of thetest compound in rabbit ocular tissues.

Solution Formulation

The compound of the invention,5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(1) was dissolved in either 10% 2-hydroxypropyl-β-cyclodextrin to attaina target concentration of 4 mg/mL or in purified water to attain atarget concentration of 1 mg/mL. Bilateral intravitreal injection (50μL/eye) of the solution of test compound was administered to New Zealandwhite rabbits in two dose groups, 200 μg/eye and 50 μg/eye,respectively, for the cyclodextrin and water vehicle formulations,respectively. The test compound concentration was measured in oculartissues: vitreous, aqueous, retina/choroid and iris-ciliary body atpre-determined time points post injection (30 min, 4 h, 1 d, 3 d, 7 d,14 d). Two rabbits (four eyes) were dosed for each time point. In thevitreous tissue, compound 1 exhibited a two-phase decrease inconcentration characterized by an initial decrease in concentration witha half-life of approximately 12 hours and finally a terminal half-lifeof approximately 3.6 days. The compound was found to distribute quicklyinto the retinal and choroidal region as well and shows a similarpharmacokinetic profile as in the vitreous tissue.

Suspension Formulation

A suspension formulation was prepared by combining crystalline compound1 of Example 1 with 0.5% hydroxypropyl methylcellulose (HPMC E5)+0.02%Tween 80 to attain a target concentration of 10 mg/mL. Bilateralintravitreal injection (50 μL/eye) of the suspension of test compoundwas administered to New Zealand white rabbits (500 μg/eye) The testcompound concentration was measured in ocular tissues as in thesuspension formulation assay at 30 min, 2 wks, 4 wks, 6 wks, and 8 wkspost injection. The compound showed a linear decrease in drugconcentration in the vitreous from 30 min to 6 weeks with a clearancerate of approximately 3 μg/mL/day. The behavior is consistent with thesolubility of compound 1 in the vehicle and the ocular pharmacokineticbehavior in the solution formulation. The drug concentration in plasmawas measured and found to be at least 3 orders of magnitude lower thanthe concentration in vitreous tissue.

Assay 9: Pharmacodynamic Assay: Inhibition of IL6-induced pSTAT3 in Rats

The ability of a single intravitreal administration of test compound toinhibit IL-6 induced pSTAT3 was measured in rat retina/choroidhomogenates.

Suspension formulations were prepared by combining crystalline compound1 of Example 1 with 0.5% hydroxypropyl methylcellulose (HPMC E5 LV),0.02% Tween 80, and 0.9% sodium chloride in purified water to attaintarget concentrations of 3 mg/mL and 10 mg/mL.

Female Lewis rats were intravitreally (IVT) dosed (5 μL per eye) withthe suspension formulations or with the drug vehicle. Three days later,IL-6 (Peprotech; 0.1 mg/mL; 5 μL per eye) or vehicle was intravitreallyadministered to induce pSTAT3. Ocular tissues were dissected one hourafter the second IVT injection with IL-6. The retina/choroid tissueswere homogenized and pSTAT3 levels were measured using an ELISA (CellSignaling Technology). The percent inhibition of IL-6-induced pSTAT3 wascalculated in comparison to the vehicle/vehicle and vehicle/IL-6 groups.Inhibition of greater than 100% reflects a reduction of pSTAT3 levels tobelow those observed in the vehicle/vehicle group.

With a 3 day pre-treatment prior to IL-6 challenge, the 15 μg dose andthe 50 μg dose of the compound of the invention administered by thesuspension formulation inhibited IL-6-induced pSTAT3 by 33% and 109%,respectively in the retina/choroid tissues.

Assay 10: Pharmacodynamic Assay: Inhibition of IFNγ-Induced IP-10 inRabbits

The ability of a single intravitreal administration of test compound toinhibit interferon-gamma (IFNγ) induced IP-10 protein levels wasmeasured in rabbit vitreous and retina/choroid tissues.

Solution formulations at concentrations of 1 mg/mL and 4 mg/mL ofcompound 1 of Example 1 were prepared as in Assay 8. A suspensionformulation was prepared by combining crystalline compound 1 of Example1 with 0.5% hydroxypropyl methylcellulose (HPMC E5), 0.02% Tween 80, and9 mg/mL sodium chloride in purified water to attain a targetconcentration of 20 mg/mL.

Male, New Zealand White rabbits (Liveon Biolabs, India) were used forthe studies. Animals were acclimated after arrival at the researchfacilities (Jubilant Biosys Ltd., India). Each rabbit was given a totalof two intravitreal (IVT) injections with a total dose volume of 50 μLper eye. The first IVT injection (45 μL per eye) delivered test compoundor vehicle at a prescribed time point (i.e. 24 hours for the solutionformulations or 1 week for the suspension formulation). The second PITinjection (5 μL per eye) delivered IFNγ (1 μg/eye; Stock solution 1mg/mL; Kingfisher Biotech) or vehicle for the induction of IP-10. Inbrief, on the day of the injections, rabbits were anesthetized with anintramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg).Once deeply anesthetized, each eye was rinsed with sterile saline andIVT injections were performed using a 0.5 mL insulin syringe (50units=0.5 mL) with a 31-gauge needle at the supra-nasal side of the botheyes by marking the position with a Braunstein fixed caliper (2¾″) 3.5mm from the rectus muscle and 4 mm from the limbus.

Tissues were collected 24 hours after the second IVT injection withIFNγ. Vitreous humor (VII) and retina/choroid tissues (R/C) werecollected and homogenized, and IP-10 protein levels were measured usinga rabbit CXCL10 (IP-10) ELISA kit (Kingfisher Biotech). The percentinhibition of IFNγ-induced IP-1.0 was calculated in comparison to thevehicle/vehicle and vehicle/IFNγ groups.

When dosed as a solution, with a 24 hour pre-treatment prior to the IFNγchallenge, 45 μg of compound 1 inhibited IFNγ-induced IP-10 by 70% and86% in the vitreous humor and retina/choroid tissue, respectively, while180 μg of the compound inhibited IFNγ-induced IP-10 by 91% and 100% inthe vitreous humor and retina/choroid tissue, respectively.

With a 1 week pre-treatment prior to the IFNγ challenge, the crystallinesuspension formulation of compound 1 inhibited IFNγ-induced IP-10 by100% in both the vitreous humor and retina/choroid tissues.

Assay 11: Pharmacokinetics in Plasma and Lung in Mouse

Plasma and lung levels of the test compound and the ratio thereof wasdetermined in the following manner. BALB/c mice from Charles RiverLaboratories were used in the assay. Test compounds were individuallyformulated in 20% propylene glycol in pH 4 citrate buffer at aconcentration of 0.2 mg/mL and 50 uL of the dosing solution wasintroduced into the trachea of a mouse by oral aspiration. At varioustime points (typically 0.167, 2, 6, 24 hr) post dosing, blood sampleswere removed via cardiac puncture and intact lungs were excised from themice. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4minutes at approximately 12,000 rpm at 4° C. to collect plasma. Lungswere padded dry, weighed, and homogenized at a dilution of 1:3 insterile water. Plasma and lung levels of test compound were determinedby LC-MS analysis against analytical standards constructed into astandard curve in the test matrix. A lung to plasma ratio was determinedas the ratio of the lung AUC in μg hr/g to the plasma AUC in μg hr/mL,where AUC is conventionally defined as the area under the curve of testcompound concentration vs. time.

The compound of the invention exhibited exposure in lung about 55 timesgreater than exposure in plasma in mouse.

Assay 12: Murine (Mouse) Model of IL-13 Induced pSTAT6 Induction in LungTissue

Il-13 is an important cytokine underlying the pathophysiology of asthma(Kudlacz et al. Eur. J Pharmacol, 2008, 582, 154-161). IL-13 binds tocell surface receptors activating members of the Janus family of kinases(JAK) which then phosphorylate STAT6 and subsequently activates furthertranscription pathways. In the described model, a dose of IL-13 wasdelivered locally into the lungs of mice to induce the phosphorylationof STAT6 (pSTAT6) which is then measured as the endpoint.

Adult balb/c mice from Harlan were used in the assay. On the day ofstudy, animals were lightly anesthetized with isoflurane andadministered either vehicle or test compound (0.5 mg/mL, 50 μL totalvolume over several breaths) via oral aspiration. Animals were placed inlateral recumbency post dose and monitored for full recovery fromanesthesia before being returned to their home cage. Four hours later,animals were once again briefly anesthetized and challenged with eithervehicle or IL-13 (0.03 μg total dose delivered, 50 μL total volume) viaoral aspiration before being monitored for recovery from anesthesia andreturned to their home cage. One hour after vehicle or IL-13administration, lungs were collected for both pSTAT6 detection using ananti-pSTAT6 ELISA (rabbit mAb capture/coating antibody; mouse mAbdetection/report antibody: anti-pSTAT6-pY641; secondary antibody:anti-mouse IgG-HRP) and analyzed for total drug concentration asdescribed above in Assay 11.

Activity in the model is evidenced by a decrease in the level of pSTAT6present in the lungs of treated animals at 5 hours compared to thevehicle treated, IL-13 challenged control animals. The differencebetween the control animals which were vehicle-treated, IL-13 challengedand the control animals which were vehicle-treated, vehicle challengeddictated the 0% and 100% inhibitory effect, respectively, in any givenexperiment. The compound of the invention exhibited about 60% inhibitionof STATE phosphorylation at 4 hours after IL-13 challenge.

Assay 13: Murine Model of Alternaria alternata-Induced EosinophilicInflammation of the Lung

Airway eosinophilia is a hallmark of human asthma. Alternaria alternatais a fungal aeroallergen that can exacerbate asthma in humans andinduces eosinophilic inflammation in the lungs of mice (Havaux et al.Clin Exp Immunol. 2005, 139(2):179-88). In mice, it has beendemonstrated that alternaria indirectly activates tissue resident type 2innate lymphoid cells in the lung, which respond to (e.g. IL-2 and IL-7)and release JAK-dependent cytokines (e.g. IL-5 and IL-13) and coordinateeosinophilic inflammation Bartemes et al. J Immunol. 2012,188(3):1503-13).

Seven- to nine-week old male C57 mice from Taconic were used in thestudy. On the day of study, animals were lightly anesthetized withisoflurane and administered either vehicle or test compound (0.1-1.0mg/mL, 50 μL total volume over several breaths) via oropharyngealaspiration. Animals were placed in lateral recumbency post dose andmonitored for full recovery from anesthesia before being returned totheir home cage. One hour later, animals were once again brieflyanesthetized and challenged with either vehicle or alternaria extract(200 ug total extract delivered, 50 μL total volume) via oropharyngealaspiration before being monitored for recovery from anesthesia andreturned to their home cage. Forty-eight hours after alternariaadministration, bronchoalveolar lavage fluid (BALF) was collected andeosinophils were counted in the BAIT using the Advia 120 HematologySystem (Siemens).

Activity in the model is evidenced by a decrease in the level ofeosinophils present in the BALF of treated animals at forty-eight hourscompared to the vehicle treated, alternaria challenged control animals.Data are expressed as percent inhibition of the vehicle treated,alternaria challenged BALF eosinophils response. To calculate percentinhibition, the number of BALF eosinophils for each condition isconverted to percent of the average vehicle treated, alternariachallenged BALF eosinophils and subtracted from one-hundred percent. Thecompound of the invention exhibited about 88% inhibition of BALFeosinophil counts at forty-eight hours after alternaria challenge.

Assay 14: Murine Model of LPS/G-CSF/IL-6/IFNγ Cocktail-Induced AirwayNeutrophilic Inflammation of the Lung Model

Airway neutrophilia is a hallmark of a range of respiratory disease inhumans. Compound 1 was tested in a model of neutrophilic airwayinflammation using a LPS/G-CSF/IL-6/IFNγ cocktail to induce airwayneutrophilia.

Seven- to nine-week old male Balb/C (wildtype) mice from JacksonLaboratory were used in the study. On the day of study, animals werelightly anesthetized with isoflurane and administered either vehicle ortest compound (1.0 mg/mL, 50 μL total volume over several breaths) viaoropharyngeal aspiration. Animals were placed in lateral recumbency postdose and monitored for full recovery from anesthesia before beingreturned to their home cage. One hour later, animals were once againbriefly anesthetized and challenged with either vehicle or LPS; 0.01mg/kg/G-CSF; 5 μg/IL-6; 5 μg/IFNγ; 5 μg (100 μL total volume) viaoropharyngeal aspiration (OA). Twenty-four hours after theLPS/G-CSF/IL-6/IFNγ cocktail administration, bronchoalveolar lavagefluid (BALF) was collected and neutrophils were counted.

Upon OA treatment with compound 1, there was a statistically significantreduction of the airway neutrophils (84% compared to vehicle treatedmice), demonstrating that the blockade of JAK-dependent signaling haseffects on neutrophilic airway inflammation.

Assay 15: Inhibition of INFγ and IL-27 Induced Chemokines CXCL9 andCXCL10 in Human 3D Airway Cultures

EpiAirway tissue cultures were obtained from Mattek (AIR-100). Cultureswere derived from asthmatic donors. In a cell culture insert, humanderived tracheal/bronchial epithelial cells were grown anddifferentiated on a porous membrane support, allowing an air-liquidinterface with warmed culture medium below the cells and a gaseous testatmosphere above. Tissues were cultured in maintenance media (Mattek,AIR-100-MM) in a 37° C., 5% CO₂ humidified incubator. Four donors weretested. On Day 0, tissue cultures were treated with test compounds at 10μM, 1 μM and/or 0.1 μM. Compounds were diluted in dimethyl sulfoxide(DMSO, Sigma) to a final concentration of 0.1%. DMSO at 0.1% was used asa vehicle control. Test compounds were incubated with cultures for 1hour at 37° C., 5% CO₂, followed by the addition of pre-warmed mediacontaining IFNγ (R&D Systems) or IL-27 (R&D Systems) at a finalconcentration at 100 ng/ml. Tissue cultures were maintained for 8 days.Media was replaced every 2 days with fresh media containing compoundsand IFNγ or IL-27. On Day 8, tissue cultures and supernatants werecollected for analysis. Supernatant samples were assayed for CXCL10(IP-10) and CXCL9 (MIG) using luminex analysis (END Millipore). Data isexpressed as % Inhibition+/−standard deviation (±STDV). Percentinhibition was determined by compound inhibitory potency against IFNγ orIL-27 induced CXCL10 or CXCL9 secretion compared to vehicle treatedcells. Data is the average from 3 or 4 donors. Compound 1 was able toinhibit IFNγ induced CXCL10 secretion by 99%±2.0 (at 10 μM), 71%±19 (atμM) and 17%±12 (at 0.1 μM) when compared to vehicle control. Compound 1was able to inhibit IFNγ induced CXCL9 secretion by 100%±0.3 (at 10 μM),99%±0.9 (at 1 μM) and 74%±17 (at 0.1 μM) when compared to vehicle.Compound 1 was able to inhibit IL-27 induced CXCL10 secretion by 108%±11(at 10 μM), 98%±10 (at 1 μM) and 73%±8.5 (at 0.1 μM) when compared tovehicle control. Compound 1 was able to inhibit IL-27 induced CXCL9secretion by 100%±0 (at 10 μM); 95%±3.7 (at 1 OA) and 75%±3.5 (at 0.1μM) when compared to vehicle control.

Assay 16: IL-5 Mediated Eosinophil Survival Assay

The potency of the test compound for IL-5 mediated eosinophil survivalwas measured in human eosinophils isolated from human whole blood(AllCells). Because IL-5 signals through JAK, this assay provides ameasure of JAK cellular potency.

Human eosinophils were isolated from fresh human whole blood (AllCells)of healthy donors. Blood was mixed with 4.5% Dextran (Sigma-Aldrich) ina 0.9% sodium chloride solution (Sigma-Aldrich). Red blood cells wereleft to sediment for 35 minutes. The leukocyte rich upper layer wasremoved and layered over Ficoll-Paque (GE Healthcare) and centrifuged at600 g for 30 minutes. The plasma and mononuclear cell layers wereremoved before the granulocyte layer was lysed with water to remove anycontaminating red blood cells. Eosinophils were further purified using ahuman eosinophil isolation kit (Miltenyi Biotec). A fraction of thepurified eosinophils were incubated with anti-CD16 FITC (MiltenyiBiotec) for 10 minutes at 4° C. in the dark. Purity was analyzed using aLSRII flow cvtorneter (BD Biosciences).

Cells were cultured in a 37° C., 5% CO₂ humidified incubator in RPMI1640 (Life Technologies) supplemented with 10% Heat Inactivated FetalBovine Serum (FBS, Life Technologies), 2 mM Glutamax (LifeTechnologies), 25 mM HEPES (Life Technologies) and 1× Pen/Strep (LifeTechnologies). Cells were seeded at 10,000 cells/well in media (50 μL).The plate was centrifuged at 300 g for 5 minutes and supernatantsremoved. Compounds were serially diluted in DMSO and then dilutedanother 500-fold to a 2× final assay concentration in media. Testcompounds (50 μL/well) were added to cells, and incubated at 37° C., 5%CO₂ for 1 hour, followed by the addition of IL-5 (R&D Systems; finalconcentrations 1 ng/mL and 10 pg/ml) in pre-warmed assay media (50 μL)for 72 hours.

After cytokine stimulation, cells were centrifuged at 300 g for 5 minand washed twice with cold DPBS (Life Technologies). To access viabilityand apoptosis, cells were incubated with Propidium Iodide (Thermo FisherScientific) and APC Annexin V (BD Biosciences) and analyzed using aLSRII flow cytometer (BD Biosciences). IC₅₀ values were determined fromanalysis of the viability curves of percent cell viability vs compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values. Compound 1 exhibited a pIC₅₀ value of 7.9±0.5 in thepresence of 10 pg/ml IL-5 and a pIC₅₀ value of 6.5±0.2 in the presenceof 1 ng/ml IL-5.

Assay 17: Pharmacodynamic Assay: Inhibition of IFNγ-Induced pSTAT1 inRabbit Eyes

The ability of a single intravitreal administration of test compound toinhibit interferon-gamma (IFNγ) induced phosphorylation of STAT1 protein(pSTAT1) was measured in rabbit retina/choroid tissue.

A suspension formulation was prepared by combining compound 1 of Example1, with 0.5% hydroxypropyl methylcellulose (HPMC E5), 0.02% Tween 80,and 9 mg/mL sodium chloride in purified water to attain a targetconcentration of 20 mg/mL.

Male, New Zealand White rabbits (Liveon Biolabs, India) were used forthe studies. Animals were acclimated after arrival at the researchfacilities (Jubilant Biosys Ltd., India). Each rabbit was given a totalof two intravitreal (IVT) injections with a total dose volume of 50 μLper eye. The first IVT injection (45 μL per eye) delivered 0.9 mg oftest compound or vehicle. One week later, a second IVT injection (5 μLper eye) delivered IFNγ (1 μg/eye; stock solution 1 mg/mL; KingfisherBiotech) or vehicle for the induction of IP-10. On the day of theinjections, rabbits were anesthetized with an intramuscular injection ofketamine (35 mg/kg) and xylazine (5 mg/kg). Once deeply anesthetized,each eye was rinsed with sterile saline and IVT injections wereperformed using a 0.5 mL insulin syringe (50 units=0.5 mL) with a31-gauge needle at the supra-nasal side of the both eyes by marking theposition with a Braunstein fixed caliper (2¾″) 3.5 mm from the rectusmuscle and 4 mm from the limbus.

Tissues were collected 2 hours after the second IVT injection with IFNγ.Retina/choroid tissues (R/C) were collected and homogenized, and pSTAT1levels were measured by quantitative Western Blot on the ProteinSimpleWES instrument. The percent inhibition of IFNγ-induced pSTAT1 wascalculated in comparison to the vehicle/vehicle and vehicle/IFNγ groups.

With a 1 week pre-treatment prior to the IFNγ challenge, the suspensionformulation of compound 1 of Example 1 inhibited IFNγ-induced pSTAT1 by85%.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A method of treating an ocular disease in amammal, the method comprising administering a pharmaceutical compositioncomprising 5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,or a pharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier to the eye of the mammal.
 2. Themethod of claim 1, wherein the ocular disease is uveitis, diabeticretinopathy, diabetic macular edema, dry eye disease, age-relatedmacular degeneration, or atopic keratoconjunctivitis.
 3. The method ofclaim 2, wherein the ocular disease is uveitis.
 4. The method of claim 1wherein the pharmaceutical composition is administered by injection. 5.The method of claim 2, wherein the ocular disease is diabeticretinopathy.
 6. The method of claim 2, wherein the ocular disease isdiabetic macular edema.
 7. The method of claim 2, wherein the oculardisease is dry eye disease.
 8. The method of claim 2, wherein the oculardisease is age-related macular degeneration.
 9. The method of claim 2,wherein the ocular disease is atopic keratoconjunctivitis.
 10. Themethod of claim 2, wherein the pharmaceutical composition is a solution.11. The method of claim 2, wherein the pharmaceutical composition is asuspension.
 12. The method of claim 2, wherein5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolis in the form of a freebase.
 13. The method of claim 12, wherein5-ethyl-2-fluoro-4-(345-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolis in the form of a crystalline hydrate.
 14. The method of claim 13,wherein the crystalline hydrate is characterized by a powder X-raydiffraction pattern comprising diffraction peaks at 2θ values of6.20±0.20, 9.58±0.20, 17.53±0.20, 19.28±0.20, and 21.51±0.2.
 15. Themethod of claim 14, wherein the powder X-ray diffraction pattern isfurther characterized by having two or more additional diffraction peaksat 2θ values selected from 10.34±0.20, 11.54±0.20, 12.77±0.20,13.01±0.20, 16.94±0.20, 20.61±0.20, and 22.10±0.20.
 16. The method ofclaim 2, wherein the pharmaceutical composition is administered byinjection.
 17. The method of claim 16, wherein the pharmaceuticalcomposition is administered by intravitreal injection.
 18. The method ofclaim 17, wherein the pharmaceutical composition is a suspension. 19.The method of claim 18, wherein the pharmaceutical composition is asuspension comprising5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolin the form of a crystalline hydrate freebase.
 20. The method of claim19, wherein the crystalline hydrate is characterized by a powder X-raydiffraction pattern comprising diffraction peaks at 2θvalues of6.20±0.20, 9.58±0.20, 17.53±0.20, 19.28±0.20, and 21.51±0.2.